Amide and Carbamate Derivatives of Alkyl Substituted N-[4-(4-Amino-1H-Imidazo[4,5-C] Quinolin-1-YL)Butyl]Methanesulfonamides and Methods

ABSTRACT

Amide and carbamate derivatives of N-[4-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamides with an ethyl, methyl, or n-propyl substituent at the 2-position, pharmaceutical compositions containing these compounds, methods of making the compounds, and methods of use of these compounds in modulating the immune system, for inducing cytokine biosyn-thesis in animals and in the treatment of diseases including viral and neoplastic diseases, are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention claims priority to U.S. Provisional ApplicationSer. No. 60/715,949, filed Sep. 9, 2005, which is incorporated herein byreference.

BACKGROUND

Certain substituted 1H-imidazo[4,5-c]pyridin-4-amine, quinolin-4-amine,tetrahydroquinolin-4-amine, naphthyridin-4-amine, andtetrahydronaphthyridin-4-amine compounds as well as certain analogousthiazolo and oxazolo compounds have been found to be useful as immuneresponse modifiers (IRMs), rendering them useful in the treatment of avariety of disorders.

There continues to be interest in and a need for compounds, theadministration of which can give rise to modulation of the immuneresponse, through induction of cytokine biosynthesis or othermechanisms.

SUMMARY OF THE INVENTION

It has now been found that certain amide and carbamate derivatives of2-alkylN-[4-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamidesgive rise to induction of cytokine biosynthesis. The present inventionprovides such compounds, which are of the following Formula I:

wherein R, R′, and Y are as defined below; and pharmaceuticallyacceptable salts thereof.

The compounds or salts of Formulas I are useful due to their ability togive rise to modulation of cytokine biosynthesis (e.g., induce thebiosynthesis or production of one or more cytokines) and otherwise bringabout modulation of the immune response when administered to animals.This makes the compounds useful in the treatment of a variety ofconditions, such as viral diseases and neoplastic diseases, that areresponsive to such changes in the immune response.

The present invention also provides pharmaceutical compositionscontaining the compounds of Formula I and methods of inducing cytokinebiosynthesis in animal cells, treating a viral disease in an animal,and/or treating a neoplastic disease in an animal by administering tothe animal one or more compounds of the Formula I, and/orpharmaceutically acceptable salts thereof, or by administering to theanimal a pharmaceutical composition containing one or more compounds ofthe Formula I, and/or pharmaceutically acceptable salts thereof.

In another aspect, the invention provides methods of synthesizing thecompounds of Formula I.

As used herein, “a,” “an,” “the,” “at least one,” and “one or more” areused interchangeably.

The terms “comprising” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. Guidance is also provided herein through listsof examples, which can be used in various combinations. In eachinstance, the recited list serves only as a representative group andshould not be interpreted as an exclusive list.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE INVENTION

The present invention provides compounds of the following Formula I:

wherein R, R′, and Y are as defined below; and pharmaceuticallyacceptable salts thereof.

In one embodiment, the present invention provides a compound of thefollowing Formula I:

wherein:

Y is selected from the group consisting of —C(O)— and —C(O)—O—;

R is selected from the group consisting of alkyl, aryl, arylalkylenyl,heteroaryl, heteroarylalkylenyl, heterocyclyl, andheterocyclylalkylenyl; wherein aryl and arylalkylenyl are unsubstitutedor substituted by one or more substituents selected from the groupconsisting of alkyl, alkoxy, aryl, and halogen; and wherein the atom inheterocyclyl attached to Y is a carbon atom; and

R′ is selected from the group consisting of methyl, ethyl, and n-propyl;or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention provides a compound of thefollowing Formula II:

wherein:

Y is selected from the group consisting of —C(O)— and —C(O)—O—; and

R is selected from the group consisting of alkyl, aryl, arylalkylenyl,heteroaryl, heteroarylalkylenyl, heterocyclyl, andheterocyclylalkylenyl; wherein aryl and arylalkylenyl are unsubstitutedor substituted by one or more substituents selected from the groupconsisting of alkyl, alkoxy, aryl, and halogen; and wherein the atom inheterocyclyl attached to Y is a carbon atom; or a pharmaceuticallyacceptable salt thereof.

In another embodiment, the present invention provides a compound of thefollowing Formula III:

wherein:

Y is selected from the group consisting of —C(O)— and —C(O)—O—; and

R is selected from the group consisting of alkyl, aryl, arylalkylenyl,heteroaryl, heteroarylalkylenyl, heterocyclyl, andheterocyclylalkylenyl; wherein aryl and arylalkylenyl are unsubstitutedor substituted by one or more substituents selected from the groupconsisting of alkyl, alkoxy, aryl, and halogen; and wherein the atom inheterocyclyl attached to Y is a carbon atom;

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention provides a compound of thefollowing Formula IV:

wherein:

Y is selected from the group consisting of —C(O)— and —C(O)—O—; and

R is selected from the group consisting of alkyl, aryl, arylalkylenyl,heteroaryl, heteroarylalkylenyl, heterocyclyl, andheterocyclylalkylenyl; wherein aryl and arylalkylenyl are unsubstitutedor substituted by one or more substituents selected from the groupconsisting of alkyl, alkoxy, aryl, and halogen; and wherein the atom inheterocyclyl attached to Y is a carbon atom;

or a pharmaceutically acceptable salt thereof.

For any of the compounds presented herein, each one of the followingvariables (e.g., R, R′, Y, and so on) in any of its embodiments can becombined with any one or more of the other variables in any of theirembodiments and associated with any one of the formulas describedherein, as would be understood by one of skill in the art. Each of theresulting combinations of variables is an embodiment of the presentinvention.

For certain embodiments, e.g., of any one of Formulas I through IV, Y isselected from the group consisting of —C(O)— and —C(O)—O—.

For certain embodiments, e.g., of any one of Formulas I through IV, Y is—C(O)—.

For certain embodiments, e.g., of any one of Formulas I through IV, Y is—C(O)—O—.

For certain embodiments, including any one of the above embodiments ofFormulas I through IV, R is selected from the group consisting of alkyl,aryl, arylalkylenyl, heteroaryl, heteroarylalkylenyl, heterocyclyl, andheterocyclylalkylenyl; wherein aryl and arylalkylenyl are unsubstitutedor substituted by one or more substituents selected from the groupconsisting of alkyl, alkoxy, aryl, and halogen; and wherein the atom inheterocyclyl attached to Y is a carbon atom.

For certain embodiments, including any one of the above embodiments ofFormulas I through IV, R is alkyl, aryl, or arylalkylenyl. For certainof these embodiments, R is C₁₋₁₀ alkyl. For certain of theseembodiments, R is C₁₋₅ alkyl. For certain of these embodiments, R isselected from the group consisting of methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, and tert-butyl.

For certain embodiments, including any one of the above embodiments ofFormulas I through IV, where not excluded, R is aryl. For certain ofthese embodiments, R is phenyl.

For certain embodiments, including any one of the above embodiments ofFormulas I through IV, where not excluded, R is arylalkylenyl. Forcertain of these embodiments, R is benzyl.

For certain embodiments, including any one of the above embodiments ofFormula I, R′ is selected from the group consisting of methyl, ethyl,and n-propyl.

For certain embodiments, including any one of the above embodiments ofFormula I, R′ is ethyl.

For certain embodiments, including any one of the above embodiments ofFormula I, where not excluded, R′ is methyl.

For certain embodiments, including any one of the above embodiments ofFormula I, where not excluded, R′ is n-propyl.

For certain embodiments, the present invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundor salt of any one of the above embodiments of Formulas I, II, III, andIV, and a pharmaceutically acceptable carrier.

For certain embodiments, the present invention provides a method ofinducing cytokine biosynthesis in an animal comprising administering aneffective amount of a compound or salt of any one of the aboveembodiments of Formulas I, II, III, and IV, or a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundor salt of any one of the above embodiments of Formulas I, II, III, andIV to the animal.

For certain embodiments, the present invention provides a method oftreating a viral disease in an animal comprising administering atherapeutically effective amount of a compound or salt of any one of theabove embodiments of Formulas I, II, III, and IV, or a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundor salt of any one of the above embodiments of Formulas I, II, III, andIV to the animal.

For certain embodiments, the present invention provides a method oftreating a neoplastic disease in an animal comprising administering atherapeutically effective amount of a compound or salt of any one of theabove embodiments of Formulas I, II, III, and IV, or a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundor salt of any one of the above embodiments of Formulas I, II, III, andIV to the animal.

As used herein, the terms “alkyl” and the prefix “alk-” are inclusive ofboth straight chain and branched chain groups and of cyclic groups,e.g., cycloalkyl and cycloalkenyl. Unless otherwise specified, thesegroups contain from 1 to 20 carbon atoms, with alkenyl groups containingfrom 2 to 20 carbon atoms, and allynyl groups containing from 2 to 20carbon atoms. In some embodiments, these groups have a total of up to 10carbon atoms, up to 8 carbon atoms, up to 6 carbon atoms, or up to 4carbon atoms. Cyclic groups can be monocyclic or polycyclic andpreferably have from 3 to 10 ring carbon atoms. Exemplary cyclic groupsinclude cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl,cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, adamantyl,and substituted and unsubstituted bornyl, norbornyl, and norbornenyl.

Unless otherwise specified, “alkylene,” is the divalent forms of the“alkyl” groups defined above. The term “alkylenyl” is used when“allylene” is substituted. For example, an arylalkylenyl group comprisesan “alkylene” moiety to which an aryl group is attached.

The term “aryl” as used herein includes carbocyclic aromatic rings orring systems. Examples of aryl groups include phenyl, naphthyl,biphenyl, fluorenyl and indenyl.

Unless otherwise indicated, the term “heteroatom” refers to the atoms O,S, or N.

The term “heteroaryl” includes aromatic rings or ring systems thatcontain at least one ring heteroatom (e.g., O, S, N). In someembodiments, the term “heteroaryl” includes a ring or ring system thatcontains 2 to 12 carbon atoms, 1 to 3 rings, 1 to 4 heteroatoms, and O,S, and/or N as the heteroatoms. Suitable heteroaryl groups includefuryl, thienyl, pyridyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl,triazolyl, pyrrolyl, tetrazolyl, imidazolyl, pyrazolyl, oxazolyl,thiazolyl, benzofuranyl, benzothiophenyl, carbazolyl, benzoxazolyl,pyrimidinyl, benzimidazolyl, quinoxalinyl, benzothiazolyl,naphthyridinyl, isoxazolyl, isothiazolyl, purinyl, quinazolinyl,pyrazinyl, 1-oxidopyridyl, pyridazinyl, triazinyl, tetrazinyl,oxadiazolyl, thiadiazolyl, and so on.

The term “heterocyclyl” includes non-aromatic rings or ring systems thatcontain at least one ring heteroatom (e.g., O, S, N) and includes all ofthe fully saturated and partially unsaturated derivatives of the abovementioned heteroaryl groups. In some embodiments, the term“heterocyclyl” includes a ring or ring system that contains 2 to 12carbon atoms, 1 to 3 rings, 1 to 4 heteroatoms, and O, S, and N as theheteroatoms. Exemplary heterocyclyl groups include pyrrolidinyl,tetrahydrofuranyl, morpholinyl, thiomorpholinyl,1,1-dioxothiomorpholinyl, piperidinyl, piperazinyl, thiazolidinyl,imidazolidinyl, isothiazolidinyl, tetrahydropyranyl, quinuclidinyl,homopiperidinyl (azepanyl), 1,4-oxazepanyl, homopiperazinyl(diazepanyl), 1,3-dioxolanyl, aziridinyl, azetidinyl,dihydroisoquinolin-(1H)-yl, octahydroisoquinolin-(1H)-yl,dihydroquinolin-(2H)-yl, octahydroquinolin-(2H)-yl,dihydro-1H-imidazolyl, 3-azabicyclo[3.2.2]non-3-yl, and the like.

The term “heterocyclyl” includes bicylic and tricyclic heterocyclic ringsystems. Such ring systems include fused and/or bridged rings and spirorings. Fused rings can include, in addition to a saturated or partiallysaturated ring, an aromatic ring, for example, a benzene ring. Spirorings include two rings joined by one spiro atom and three rings joinedby two spiro atoms.

When “heterocyclyl” contains a nitrogen atom, the point of attachment ofthe heterocyclyl group may be the nitrogen atom unless otherwisespecified.

The invention is inclusive of the compounds described herein (includingintermediates) in any of their pharmaceutically acceptable forms,including isomers (e.g., diastereomers and enantiomers), salts,solvates, polymorphs, and the like. In particular, if a compound isoptically active, the invention specifically includes each of thecompound's enantiomers as well as racemic and scalemic mixtures of theenantiomers. It should be understood that the term “compound” includesany or all of such forms, whether explicitly stated or not (although attimes, “salts” are explicitly stated).

Preparation of the Compounds

Compounds of the invention may be synthesized by synthetic routes thatinclude processes analogous to those well known in the chemical arts,particularly in light of the description contained herein. The reagentsare generally available from commercial sources such as AldrichChemicals (Milwaukee, Wis., USA) or are readily prepared using methodswell known to those skilled in the art (e.g., prepared by methodsgenerally described in Louis F. Fieser and Mary Fieser, Reagents forOrganic Synthesis, v. 1-19, Wiley, New York, (1967-1999 ed.); Alan R.Katritsky, Otto Meth-Cohn, Charles W. Rees, Comprehensive OrganicFunctional Group Transformations, v 1-6, Pergamon Press, Oxford,England, (1995); Barry M. Trost and Ian Fleming, Comprehensive OrganicSynthesis, v. 1-8, Pergamon Press, Oxford, England, (1991); orBeilsteins Handbuch der organischen Chemie, 4, Aufl. Ed.Springer-Verlag, Berlin, Germany, including supplements (also availablevia the Beilstein online database)).

For illustrative purposes, the reaction schemes depicted below providepotential routes for synthesizing the compounds of the presentinvention. For more detailed description of the individual reactionsteps, see the EXAMPLES section below. Those skilled in the art willappreciate that other synthetic routes may be used to synthesize thecompounds of the invention.

Conventional methods and techniques of separation and purification canbe used to isolate compounds of the invention, as well as various,pharmaceutically acceptable salts thereof. Such techniques may include,for example, all types of chromatography (high performance liquidchromatography (HPLC), column chromatography using common absorbentssuch as silica gel, and thin layer chromatography), recrystallization,and differential (i.e., liquid-liquid) extraction techniques.

Compounds of the invention can be prepared according to Reaction SchemeI wherein R and R′ are as defined above. In Reaction Scheme I a1H-imidazo[4,5-c]quinolin-4-amine of Formula V is reacted with an acidanhydride of Formula VI to provide aN-(1H-imidazo[4,5-c]quinolin-4-yl)amide of Formula VII which is asubgenus of Formulas I, II, III, and IV. The reaction is carried out bycombining a 1H-imidazo[4,5-c]quinolin-4-amine of Formula V with an acidanhydride of Formula VI in a suitable solvent such asN-methyl-2-pyrrolidinone. The reaction can be carried out at ambienttemperature and the product or a pharmaceutically acceptable saltthereof can be isolated using conventional methods.1H-imidazo[4,5-c]quinolin-4-amines of Formula V are known and can beprepared using known synthetic methods, see U.S. Pat. No. 6,677,349 andthe documents cited therein.

Compounds of the invention can be prepared according to Reaction SchemeII wherein R and R′ are as defined above. In Reaction Scheme II a1H-imidazo[4,5-c]quinolin-4-amine of Formula V is reacted with achloroformate of Formula VIII to provide a1H-imidazo[4,5-c]quinolin-4-ylcarbamate of Formula IX which is asubgenus of Formulas I, II, III, and IV. The reaction is carried out byadding a chloroformate of Formula VIII in a controlled fashion to asuspension or solution of a 1H-imidazo[4,5-c]quinolin-4-amine of FormulaV in a suitable solvent such as N,N-dimethylformamide orN-methyl-2-pyrrolidinone in the presence of a base such astriethylamine. The addition can be carried out at a sub-ambienttemperature, such as for example 0° C. The product or a pharmaceuticallyacceptable salt thereof can be isolated using conventional methods.

Compounds of the invention can be prepared according to Reaction SchemeIII wherein R and R′ are as defined above. In Reaction Scheme III a1H-imidazo[4,5-c]quinolin-4-amine of Formula V is reacted with an acidchloride of Formula X to provide aN-(1H-imidazo[4,5-c]quinolin-4-yl)amide of Formula VII which is asubgenus of Formulas I, II, III, and IV. The reaction is carried out bycombining a 1H-imidazo[4,5-c]quinolin-4-amine of Formula V with an acidchloride of Formula VI in a suitable solvent such as dichloromethane,N,N-dimethylformamide or N-methyl-2-pyrrolidinone in the presence of abase such as triethylamine. The reaction can be carried out at ambienttemperature and the product or a pharmaceutically acceptable saltthereof can be isolated using conventional methods.

Pharmaceutical Compositions and Biological Activity

Pharmaceutical compositions of the invention contain a therapeuticallyeffective amount of a compound or salt described above in combinationwith a pharmaceutically acceptable carrier.

The terms “a therapeutically effective amount” and “effective amount”mean an amount of the compound or salt sufficient to induce atherapeutic or prophylactic effect, such as cytokine induction,immunomodulation, antitumor activity, and/or antiviral activity. Theexact amount of compound or salt used in a pharmaceutical composition ofthe invention will vary according to factors known to those of skill inthe art, such as the physical and chemical nature of the compound orsalt, the nature of the carrier, and the intended dosing regimen.

In some embodiments, the compositions of the invention will containsufficient active ingredient or prodrug to provide a dose of about 100nanograms per kilogram (ng/kg) to about 50 milligrams per kilogram(mg/kg), preferably about 10 micrograms per kilogram (μg/kg) to about 5mg/kg, of the compound or salt to the subject.

In other embodiments, the compositions of the invention will containsufficient active ingredient or prodrug to provide a dose of, forexample, from about 0.01 mg/m² to about 5.0 mg/m², computed according tothe Dubois method, in which the body surface area of a subject (m²) iscomputed using the subject's body weight: m²=(wt kg^(0.425)×heightcm^(0.725))×0.007184, although in some embodiments the methods may beperformed by administering a compound or salt or composition in a doseoutside this range. In some of these embodiments, the method includesadministering sufficient compound to provide a dose of from about 0.1mg/m² to about 2.0 mg/m² to the subject, for example, a dose of fromabout 0.4 mg/m² to about 1.2 mg/m².

A variety of dosage forms may be used, such as tablets, lozenges,capsules, parenteral formulations, syrups, creams, ointments, aerosolformulations, transdermal patches, transmucosal patches and the like.These dosage forms can be prepared with conventional pharmaceuticallyacceptable carriers and additives using conventional methods, whichgenerally include the step of bringing the active ingredient intoassociation with the carrier.

The compounds or salts of the invention can be administered as thesingle therapeutic agent in the treatment regimen, or the compounds orsalts described herein may be administered in combination with oneanother or with other active agents, including additional immuneresponse modifiers, antivirals, antibiotics, antibodies, proteins,peptides, oligonucleotides, etc.

Compounds or salts of the invention have been shown to induce theproduction of certain cytokines in experiments performed according tothe tests set forth below. These results indicate that the compounds orsalts are useful for modulating the immune response in a number ofdifferent ways, rendering them useful in the treatment of a variety ofdisorders.

Cytokines whose production may be induced by the administration ofcompounds or salts of the invention generally include interferon-α(IFN-α) and tumor necrosis factor-α (TNF-α) as well as certaininterleukins (IL). Cytokines whose biosynthesis may be induced bycompounds or salts of the invention include IFN-α, TNF-α, IL-1, IL-6,IL-10 and IL-12, and a variety of other cytokines. Among other effects,these and other cytokines can inhibit virus production and tumor cellgrowth, making the compounds or salts useful in the treatment of viraldiseases and neoplastic diseases. Accordingly, the invention provides amethod of inducing cytokine biosynthesis in an animal comprisingadministering an effective amount of a compound or salt of the inventionto the animal. The animal to which the compound or salt is administeredfor induction of cytokine biosynthesis may have a disease as describedinfra, for example a viral disease or a neoplastic disease, andadministration of the compound or salt may provide therapeutictreatment. Alternatively, the compound or salt may be administered tothe animal prior to the animal acquiring the disease so thatadministration of the compound or salt may provide a prophylactictreatment.

In addition to the ability to induce the production of cytokines,compounds or salts described herein can affect other aspects of theinnate immune response. For example, natural killer cell activity may bestimulated, an effect that may be due to cytokine induction. Thecompounds or salts may also activate macrophages, which in turnstimulate secretion of nitric oxide and the production of additionalcytokines. Further, the compounds or salts may cause proliferation anddifferentiation of B-lymphocytes.

Compounds or salts described herein can also have an effect on theacquired immune response. For example, the production of the T helpertype 1 (T_(H)1) cytokine IFN-γ may be induced indirectly and theproduction of the T helper type 2 (T_(H)2) cytokines IL-4, IL-5 andIL-13 may be inhibited upon administration of the compounds or salts.

Whether for prophylaxis or therapeutic treatment of a disease, andwhether for effecting innate or acquired immunity, the compound or saltor composition may be administered alone or in combination with one ormore active components as in, for example, a vaccine adjuvant. Whenadministered with other components, the compound or salt or compositionand other component or components may be administered separately;together but independently such as in a solution; or together andassociated with one another such as (a) covalently linked or (b)non-covalently associated, e.g., in a colloidal suspension.

Conditions for which compounds or salts or compositions identifiedherein may be used as treatments include, but are not limited to:

(a) viral diseases such as, for example, diseases resulting frominfection by an adenovirus, a herpesvirus (e.g., HSV-I, HSV-II, CMV, orVZV), a poxvirus (e.g., an orthopoxvirus such as variola or vaccinia, ormolluscum contagiosum), a picornavirus (e.g., rhinovirus orenterovirus), an orthomyxovirus (e.g., influenzavirus), a paramyxovirus(e.g., parainfluenzavirus, mumps virus, measles virus, and respiratorysyncytial virus (RSV)), a coronavirus (e.g., SARS), a papovavirus (e.g.,papillomaviruses, such as those that cause genital warts, common warts,or plantar warts), a hepadnavirus (e.g., hepatitis B virus), aflavivirus (e.g., hepatitis C virus or Dengue virus), or a retrovirus(e.g., a lentivirus such as HIV);

(b) bacterial diseases such as, for example, diseases resulting frominfection by bacteria of, for example, the genus Escherichia,Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria,Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas,Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria,Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter,Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia,Haemophilus, or Bordetella;

(c) other infectious diseases, such as chlamydia, fungal diseasesincluding but not limited to candidiasis, aspergillosis, histoplasmosis,cryptococcal meningitis, or parasitic diseases including but not limitedto malaria, pneumocystis carnii pneumonia, leishmaniasis,cryptosporidiosis, toxoplasmosis, and trypanosome infection;

(d) neoplastic diseases, such as intraepithelial neoplasias, cervicaldysplasia, actinic keratosis, basal cell carcinoma, squamous cellcarcinoma, renal cell carcinoma, Kaposi's sarcoma, melanoma, leukemiasincluding but not limited to acute myeloid leukemia, acute lymphocyticleukemia, chronic myeloid leukemia, chronic lymphocytic leukemia,multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneousT-cell lymphoma, B-cell lymphoma, and hairy cell leukemia, and othercancers;

(e) T_(H)2-mediated, atopic diseases, such as atopic dermatitis oreczema, eosinophilia, asthma, allergy, allergic rhinitis, and Ommen'ssyndrome;

(f) certain autoimmune diseases such as systemic lupus erythematosus,essential thrombocythaemia, multiple sclerosis, discoid lupus, alopeciaareata; and

(g) diseases associated with wound repair such as, for example,inhibition of keloid formation and other types of scarring (e.g.,enhancing wound healing, including chronic wounds).

Additionally, a compound or salt identified herein may be useful as avaccine adjuvant for use in conjunction with any material that raiseseither humoral and/or cell mediated immune response, such as, forexample, live viral, bacterial, or parasitic immunogens; inactivatedviral, tumor-derived, protozoal, organism-derived, fungal, or bacterialimmunogens; toxoids; toxins; self-antigens; polysaccharides; proteins;glycoproteins; peptides; cellular vaccines; DNA vaccines; autologousvaccines; recombinant proteins; and the like, for use in connectionwith, for example, BCG, cholera, plague, typhoid, hepatitis A, hepatitisB, hepatitis C, influenza A, influenza B, parainfluenza, polio, rabies,measles, mumps, rubella, yellow fever, tetanus, diphtheria, hemophilusinfluenza b, tuberculosis, meningococcal and pneumococcal vaccines,adenovirus, HIV, chicken pox, cytomegalovirus, dengue, feline leukemia,fowl plague, HSV-1 and HSV-2, hog cholera, Japanese encephalitis,respiratory syncytial virus, rotavirus, papilloma virus, yellow fever,and Alzheimer's Disease.

Compounds or salts identified herein may be particularly helpful inindividuals having compromised immune function. For example, compoundsor salts may be used for treating the opportunistic infections andtumors that occur after suppression of cell mediated immunity in, forexample, transplant patients, cancer patients and HIV patients.

Thus, one or more of the above diseases or types of diseases, forexample, a viral disease or a neoplastic disease may be treated in ananimal in need thereof (having the disease) by administering atherapeutically effective amount of a compound or salt of the inventionto the animal.

An animal may also be vaccinated by administering an effective amount ofa compound or salt described herein, as a vaccine adjuvant. In oneembodiment, there is provided a method of vaccinating an animalcomprising administering an effective amount of a compound or saltdescribed herein to the animal as a vaccine adjuvant.

An amount of a compound or salt effective to induce cytokinebiosynthesis is an amount sufficient to cause one or more cell types,such as monocytes, macrophages, dendritic cells and B-cells to producean amount of one or more cytokines such as, for example, IFN-α, TNF-α,IL-1, IL-6, IL-10 and IL-12 that is increased (induced) over abackground level of such cytokines. The precise amount will varyaccording to factors known in the art but is expected to be a dose ofabout 100 ng/kg to about 50 mg/kg, preferably about 10 μg/kg to about 5mg/kg. In other embodiments, the amount is expected to be a dose of, forexample, from about 0.01 mg/m² to about 5.0 mg/m², (computed accordingto the Dubois method as described above) although in some embodimentsthe induction or inhibition of cytokine biosynthesis may be performed byadministering a compound or salt in a dose outside this range. In someof these embodiments, the method includes administering sufficientcompound or salt or composition to provide a dose of from about 0.1mg/m² to about 2.0 mg/m² to the subject, for example, a dose of fromabout 0.4 mg/m² to about 1.2 mg/m².

The invention also provides a method of treating a viral infection in ananimal and a method of treating a neoplastic disease in an animalcomprising administering an effective amount of a compound or salt ofthe invention to the animal. An amount effective to treat or inhibit aviral infection is an amount that will cause a reduction in one or moreof the manifestations of viral infection, such as viral lesions, viralload, rate of virus production, and mortality as compared to untreatedcontrol animals. The precise amount that is effective for such treatmentwill vary according to factors known in the art but is expected to be adose of about 100 ng/kg to about 50 mg/kg, preferably about 10 μg/kg toabout 5 mg/kg. An amount of a compound or salt effective to treat aneoplastic condition is an amount that will cause a reduction in tumorsize or in the number of tumor foci. Again, the precise amount will varyaccording to factors known in the art but is expected to be a dose ofabout 100 ng/kg to about 50 mg/kg, preferably about 10 μg/kg to about 5mg/kg. In other embodiments, the amount is expected to be a dose of, forexample, from about 0.01 mg/m² to about 5.0 mg/m², (computed accordingto the Dubois method as described above) although in some embodimentseither of these methods may be performed by administering a compound orsalt in a dose outside this range. In some of these embodiments, themethod includes administering sufficient compound or salt to provide adose of from about 0.1 mg/m² to about 2.0 mg/m² to the subject, forexample, a dose of from about 0.4 mg/m² to about 1.2 mg/m².

Thus, one or more of the above diseases or types of diseases, forexample, a viral disease or a neoplastic disease may be treated in ananimal in need thereof (having the disease) by administering atherapeutically effective amount of a compound or salt of Formula I, II,III, IV, any of the embodiments described herein, or a combinationthereof to the animal. An animal may also be vaccinated by administeringan effective amount of a compound or salt of Formula I, II, III, IV, anyof the embodiments described herein, or a combination thereof to theanimal as a vaccine adjuvant. In one embodiment, there is provided amethod of vaccinating an animal comprising administering an effectiveamount of a compound or salt described herein to the animal as a vaccineadjuvant.

The methods of the invention may be performed on any suitable subject.Suitable subjects include but are not limited to animals such as but notlimited to humans, non-human primates, rodents, dogs, cats, horses,pigs, sheep, goats, or cows.

In addition to the formulations and uses described specifically herein,other formulations, uses, and administration devices suitable forcompounds of the present invention are described in, for example,International Publication Nos. WO 03/077944 and WO 02/036592, U.S. Pat.No. 6,245,776, and U.S. Publication Nos. 2003/0139364, 2003/185835,2004/0258698, 2004/0265351, 2004/076633, and 2005/0009858.

Objects and advantages of this invention are further illustrated by thefollowing examples, but the particular materials and amounts thereofrecited in these examples, as well as other conditions and details,should not be construed to unduly limit this invention.

EXAMPLES

In the examples below automated flash chromatography was carried outusing a COMBIFLASH system (an automated high-performance flashpurification product available from Teledyne Isco, Inc., Lincoln, Nebr.,USA) or a HORIZON HPFC system (an automated high-performance flashpurification product available from Biotage, Inc, Charlottesville, Va.,USA). The eluent used for each purification is given in the example. Insome chromatographic separations, the solvent mixture 80/18/2 v/v/vchloroform/methanol/concentrated ammonium hydroxide (CMA) was used asthe polar component of the eluent. In these separations, CMA was mixedwith chloroform in the indicated ratio.

Example 1N-(2-Ethyl-1-{4-[(methylsulfonyl)amino]butyl}-1H-imidazo[4,5-c]quinolin-4-yl)acetamide

Acetic anhydride (10 mL) and N-methyl-2-pyrrolidinone (NMP) (50 mL) wereadded sequentially toN-[4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide(0.5 g) and the reaction mixture was stirred for 1 hour. The reactionmixture was diluted with ethyl acetate (about 550 mL) and then washedwith water (1 L total in portions of 100-150 mL). The organic layer wasseparated, dried over magnesium sulfate, filtered, and then concentratedunder reduced pressure. The residue was purified by automated flashchromatography (silica gel eluting with a gradient of 0-10% methanol indichloromethane containing 1% ammonium hydroxide) to provide about 100mg of an oil. The oil was dissolved in acetonitrile. Diethyl ether wasadded to the solution and it was concentrated under reduced pressure toprovide 100 mg ofN-(2-ethyl-1-{4-[(methylsulfonyl)amino]butyl})-1H-imidazo[4,5-c]quinolin-4-yl)acetamideas a white solid, mp 90-91° C. Anal calcd for C₁₉H₂₅N₅O₃S: C, 56.55; H,6.24; N, 17.36. Found: C, 56.12; H, 5.95; N, 16.98.

Example 2N-(2-Ethyl-1-{4-[(methylsulfonyl)amino]butyl}-1H-imidazo[4,5-c]quinolin-4-yl)benzamide

NMP (5 mL) and benzoic anhydride (0.63 g, 1 equivalent (eq)) were addedsequentially toN-[4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide(1 g, 1 eq) and the reaction mixture was stirred at ambient temperature.After 4 hours additional NMP (about 1 mL) was added. After 5 hoursadditional benzoic anhydride (0.63 g) was added and the reaction mixturewas stirred overnight. The reaction mixture was diluted with diethylether (about 50 mL). A white solid was isolated by filtration, rinsedwith diethyl ether, and then dried under vacuum to provide about 1 g ofa white solid. This material was purified by automated flashchromatography (silica gel eluting with a gradient of 0-10% methanol indichloromethane containing 1% ammonium hydroxide) to provide 507 mg ofN-(2-ethyl-1-{4-[(methylsulfonyl)amino]butyl}-1H-imidazo[4,5-c]quinolin-4-yl)benzamideas a white powder, mp 249-250° C. Anal calcd for C₂₄H₂₇N₅O₃S.0.10H₂O: C,61.68; H, 5.87; N, 14.98. Found: C, 61.42; H, 5.80; N, 14.96.

Example 3 Ethyl2-Ethyl-1-(4-[(methylsulfonyl)amino]butyl}-1H-imidazo[4,5-c]quinolin-4-ylcarbamate

Triethylamine (3.86 mL, 10 eq) was added to a chilled (ice/water bath)suspension ofN-[4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide(1.0 g, 1 eq) in N,N-dimethylformamide (DMF) (10 mL). Ethylchloroformate (1.5 g, 5 eq) was added dropwise to give a clear solution.The reaction mixture was allowed to come to ambient temperature withstirring for 2 hours. Additional triethylamine (3.86 mL, 10 eq) andethyl chloroformate (3.86 mL, 10 eq) were added and the reaction mixturewas stirred overnight. The reaction mixture was poured onto crushed iceand then extracted with dichloromethane (50 mL). The organic layer waswashed sequentially with water (2×50 mL), 4% sodium carbonate (50 mL),water (50 mL), and brine (50 mL) and then concentrated under reducedpressure. The residue was purified by automated flash chromatography(silica gel eluted with a linear gradient of 0-25% CMA in chloroform,2500 mL) followed by recrystallization from acetonitrile to provide0.815 g of ethyl2-ethyl-1-{4-[(methylsulfonyl)amino]butyl}-1H-imidazo[4,5-c]quinolin-4-ylcarbamateas a white crystalline solid, mp 185-187° C. Anal calcd for C₂₀H₂₇N₅O₄S:C, 55.41; H, 6.28; N, 16.15. Found: C, 55.30; H, 6.29; N, 16.24.

Example 4N-(2-Ethyl-1-{4-[(methylsulfonyl)amino]butyl}-1H-imidazo[4,5-c]quinolin-4-yl)-2-methylpropanamide

NMP (3 mL) and isobutyric anhydride (459 μL, 1 eq) were addedsequentially toN-[4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide(1 g, 1 eq) and the reaction mixture was stirred at ambient temperature.After 4 hours additional isobutyric anhydride (about 0.5 eq) was addedand the reaction mixture was stirred overnight. The reaction mixture wasdiluted with diethyl ether (about 25 mL) and stirred for 15 minutes. Thesolvent was decanted from the resulting white precipitate. The solid wasdried under vacuum and then purified by automated flash chromatography(silica gel eluting with a gradient of 0-10% methanol in dichloromethanecontaining 1% ammonium hydroxide) to provide 1.1 g ofN-(2-ethyl-1-{4-[(methylsulfonyl)amino]butyl}-1H-imidazo[4,5-c]quinolin-4-yl)-2-methylpropanamideas a white powder, mp 122-124° C. Anal calcd for C₂₁H₂₉N₅O₃S: C, 58.45;H, 6.77; N, 16.23. Found: C, 57.69; H, 6.82; N, 16.06.

Example 5N-(2-Ethyl-1-{4-[(methylsulfonyl)amino]butyl}-1H-imidazo[4,5-c]quinolin-4-yl)-2,2-dimethylpropanamide

NMP (3 mL) and trimethylacetic anhydride (564 μL, 1 eq) were addedsequentially toN-[4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide(1 g, 1 eq) and the reaction mixture was stirred at ambient temperature.After 4 hours additional trimethylacetic anhydride (about 0.5 eq) wasadded and the reaction mixture was stirred overnight. The reactionmixture was diluted with diethyl ether (about 25 mL) and stirred for 15minutes. The solvent was decanted from the resulting white precipitate.The solid was dried under vacuum and then purified by automated flashchromatography (silica gel eluting with a gradient of 0-10% methanol indichloromethane containing 1% ammonium hydroxide) to provide 0.822 g ofN-(2-ethyl-1-{4-[(methylsulfonyl)amino]butyl}-1H-imidazo[4,5-c]quinolin-4-yl)-2,2-dimethylpropanamideas a white powder, mp 220-221° C. Anal calcd for C₂₂H₃₁N₅O₃S: C, 59.30;H, 7.01; N, 15.72. Found: C, 59.44; H, 6.90; N, 15.87.

Example 6 Propyl2-Ethyl-1-{4-[(methylsulfonyl)amino]butyl}-1H-imidazo[4,5-c]quinolin-4-ylcarbamate

Triethylamine (1.93 mL, 5 eq) was added to a chilled (ice/water bath)suspension ofN-[4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide(1.0 g, 1 eq) in NMP (6.0 mL). Propyl chloroformate (1.02 g, 3 eq) wasadded dropwise. The reaction mixture was allowed to come to ambienttemperature and stirred for 24 hours. The reaction mixture was dilutedwith cold water (25 mL) and then extracted with dichloromethane (50 mL).The organic layer was washed sequentially with water (2×50 mL), 4%sodium carbonate (50 mL), water (50 mL), and brine (50 mL) and thenconcentrated under reduced pressure. The residue was purified byautomated flash chromatography (silica gel eluted with a linear gradientof 0-25% CMA in chloroform, 2000 mL) followed by recrystallization fromacetonitrile to provide 0.427 g of propyl2-ethyl-1-{4-[(methylsulfonyl)amino]butyl}-1H-imidazo[4,5-c]quinolin-4-ylcarbamateas a white solid, mp 155-157° C. Anal calcd for C₂₁H₂₉N₅O₄S: C, 56.36;H, 6.53; N, 15.65. Found: C, 56.37; H, 6.37; N, 15.70.

Example 7 Butyl2-Ethyl-1-{4-[(methylsulfonyl)amino]butyl}-1H-imidazo[4,5-c]quinolin-4-ylcarbamate

Triethylamine (1.93 mL, 5 eq) was added to a chilled (ice/water bath)suspension ofN-[4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide(1.0 g, 1 eq) in NMP (6.0 mL). n-Butyl chloroformate (1.13 g, 3 eq) wasadded dropwise. The reaction mixture was allowed to come to ambienttemperature and stirred for 24 hours. The reaction mixture was dilutedwith cold water (25 mL) and then extracted with dichloromethane (50 mL).The organic layer was washed sequentially with water (2×50 mL), 4%sodium carbonate (50 mL), water (50 mL), and brine (50 mL) and thenconcentrated under reduced pressure. The residue was purified byautomated flash chromatography (silica gel eluted with a linear gradientof 0-25% CMA in chloroform, 2000 mL) followed by recrystallization fromacetonitrile to provide 0.35 g of butyl2-ethyl-1-{4-[(methylsulfonyl)amino]butyl}-1H-imidazo[4,5-c]quinolin-4-ylcarbamateas a white solid, mp 164-166° C. Anal calcd for C₂₂H₃₁N₅O₄S: C, 57.25;H, 6.77; N, 15.17. Found: C, 57.13; H, 6.61; N, 15.20.

Exemplary Compounds

Certain exemplary compounds, including some of those described above inthe Examples, have one of the following Formulas (IIa, IIIa, or IVa) anda —Y—R substituent shown in the following table, wherein each line ofthe table is matched with a Formula (IIa, IIIa, or IVa) to represent aspecific embodiment of the invention.

—Y—R —C(O)—CH₃ —C(O)—CH₂CH₃ —C(O)—CH₂CH₂CH₃ —C(O)—CH₂(CH₃)₂—C(O)—CH₂CH₂CH₂CH₃ —C(O)—CH₂CH₂(CH₃)₂ —C(O)—C(CH₃)₃ —C(O)—phenyl—C(O)—CH₂—phenyl —C(O)—O—CH₃ —C(O)—O—CH₂CH₃ —C(O)—O—CH₂CH₂CH₃—C(O)—O—CH₂(CH₃)₂ —C(O)—O—CH₂CH₂CH₂CH₃ —C(O)—O—CH₂CH₂(CH₃)₂—C(O)—O—C(CH₃)₃ —C(O)—O—phenyl —C(O)—O—CH₂—phenyl

Compounds of the invention have been found to bring about modulation ofcytokine biosynthesis as shown by increased levels of interferon αand/or tumor necrosis factor α in human cells when tested using themethod described below.

Cytokine Induction in Human Cells

An in vitro human blood cell system is used to assess cytokineinduction. Activity is based on the measurement of interferon (α) andtumor necrosis factor (α) (IFN-α and TNF-α, respectively) secreted intoculture media as described by Testerman et al. in “Cytokine Induction bythe Immunomodulators Imiquimod and S-27609”, Journal of LeukocyteBiology, 58, 365-372 (September, 1995).

Blood Cell Preparation for Culture

Whole blood from healthy human donors is collected by venipuncture intovacutainer tubes or syringes containing EDTA. Peripheral bloodmononuclear cells (PBMC) are separated from whole blood by densitygradient centrifugation using HISTOPAQUE-1077 (Sigma, St. Louis, Mo.) orFicoll-Paque Plus (Amersham Biosciences Piscataway, N.J.). Blood isdiluted 1:1 with Dulbecco's Phosphate Buffered Saline (DPBS) or Hank'sBalanced Salts Solution (HBSS). Alternately, whole blood is placed inAccuspin (Sigma) or LeucoSep (Greiner Bio-One, Inc., Longwood, Fla.)centrifuge frit tubes containing density gradient medium. The PBMC layeris collected and washed twice with DPBS or HBSS and re-suspended at4×10⁶ cells/mL in RPMI complete. The PBMC suspension is added to 96 wellflat bottom sterile tissue culture plates containing an equal volume ofRPMI complete media containing test compound.

Compound Preparation

The compounds are solubilized in dimethyl sulfoxide (DMSO). The DMSOconcentration should not exceed a final concentration of 1% for additionto the culture wells. The compounds are generally tested atconcentrations ranging from 30-0.014 μM. Controls include cell sampleswith media only, cell samples with DMSO only (no compound), and cellsamples with reference compound.

Incubation

The solution of test compound is added at 60 μM to the first wellcontaining RPMI complete and serial 3 fold dilutions are made in thewells. The PBMC suspension is then added to the wells in an equalvolume, bringing the test compound concentrations to the desired range(usually 30-0.014 μM). The final concentration of PBMC suspension is2×10⁶ cells/mL. The plates are covered with sterile plastic lids, mixedgently and then incubated for 18 to 24 hours at 37° C. in a 5% carbondioxide atmosphere.

Separation

Following incubation the plates are centrifuged for 10 minutes at 1000rpm (approximately 200×g) at 4° C. The cell-free culture supernatant isremoved and transferred to sterile polypropylene tubes. Samples aremaintained at −30 to −70° C. until analysis. The samples are analyzedfor IFN-α by ELISA and for TNF-α by IGEN/BioVeris Assay.

Interferon (α) and Tumor Necrosis Factor (α) Analysis

IFN-α concentration is determined with a human multi-subtypecolorimetric sandwich ELISA (Catalog Number 41105) from PBL BiomedicalLaboratories, Piscataway, N.J. Results are expressed in pg/mL.

The TNF-α concentration is determined by ORIGEN M-Series Immunoassay andread on an IGEN M-8 analyzer from BioVeris Corporation, formerly knownas IGEN International, Gaithersburg, Md. The immunoassay uses a humanTNF-α capture and detection antibody pair (Catalog Numbers AHC3419 andAHC3712) from Biosource International, Camarillo, Calif. Results areexpressed in pg/mL.

Assay Data and Analysis

In total, the data output of the assay consists of concentration valuesof TNF-α and IFN-α (y-axis) as a function of compound concentration(x-axis).

Analysis of the data has two steps. First, the greater of the mean DMSO(DMSO control wells) or the experimental background (usually 20 pg/mLfor IFN-α and 40 pg/mL for TNF-α) is subtracted from each reading. Ifany negative values result from background subtraction, the reading isreported as “*”, and is noted as not reliably detectable. In subsequentcalculations and statistics, “*”, is treated as a zero. Second, allbackground subtracted values are multiplied by a single adjustment ratioto decrease experiment to experiment variability. The adjustment ratiois the area of the reference compound in the new experiment divided bythe expected area of the reference compound based on the past 61experiments (unadjusted readings). This results in the scaling of thereading (y-axis) for the new data without changing the shape of thedose-response curve. The reference compound used is2-[4-amino-2-ethoxymethyl-6,7,8,9-tetrahydro-α,α-dimethyl-1H-imidazo[4,5-c]quinolin-1-yl]ethanolhydrate (U.S. Pat. No. 5,352,784; Example 91) and the expected area isthe sum of the median dose values from the past 61 experiments.

The minimum effective concentration is calculated based on thebackground-subtracted, reference-adjusted results for a given experimentand compound. The minimum effective concentration (μmolar) is the lowestof the tested compound concentrations that induces a response over afixed cytokine concentration for the tested cytokine (usually 20 pg/mLfor IFN-α and 40 pg/mL for TNF-α). The maximal response is the maximalamount of cytokine (pg/ml) produced in the dose-response.

Cytokine Induction in Human Cells High Throughput Screen

The CYTOKINE INDUCTION IN HUMAN CELLS test method described above wasmodified as follows for high throughput screening.

Blood Cell Preparation for Culture

Whole blood from healthy human donors is collected by venipuncture intovacutainer tubes or syringes containing EDTA. Peripheral bloodmononuclear cells (PBMC) are separated from whole blood by densitygradient centrifugation using HISTOPAQUE-1077 (Sigma, St. Louis, Mo.) orFicoll-Paque Plus (Amersham Biosciences Piscataway, N.J.). Whole bloodis placed in Accuspin (Sigma) or LeucoSep (Greiner Bio-One, Inc.,Longwood, Fla.) centrifuge frit tubes containing density gradientmedium. The PBMC layer is collected and washed twice with DPBS or HBSSand re-suspended at 4×10⁶ cells/mL in RPMI complete (2-fold the finalcell density). The PBMC suspension is added to 96-well flat bottomsterile tissue culture plates.

Compound Preparation

The compounds are solubilized in dimethyl sulfoxide (DMSO). Thecompounds are generally tested at concentrations ranging from 30-0.014μM. Controls include cell samples with media only, cell samples withDMSO only (no compound), and cell samples with a reference compound2-[4-amino-2-ethoxymethyl-6,7,8,9-tetrahydro-α,α-dimethyl-1H-imidazo[4,5-c]quinolin-1-yl]ethanolhydrate (U.S. Pat. No. 5,352,784; Example 91) on each plate. Thesolution of test compound is added at 7.5 mM to the first well of adosing plate and serial 3 fold dilutions are made for the 7 subsequentconcentrations in DMSO. RPMI Complete media is then added to the testcompound dilutions in order to reach a final compound concentration of2-fold higher (60-0.028 μM) than the final tested concentration range.

Incubation

Compound solution is then added to the wells containing the PBMCsuspension bringing the test compound concentrations to the desiredrange (usually 30-0.014 μM) and the DMSO concentration to 0.4%. Thefinal concentration of PBMC suspension is 2×10⁶ cells/mL. The plates arecovered with sterile plastic lids, mixed gently and then incubated for18 to 24 hours at 37° C. in a 5% carbon dioxide atmosphere.

Separation

Following incubation the plates are centrifuged for 10 minutes at 1000rpm (approximately 200 g) at 4° C. 4-plex Human Panel MSD MULTI-SPOT96-well plates are pre-coated with the appropriate capture antibodies byMesoScale Discovery, Inc. (MSD, Gaithersburg, Md.). The cell-freeculture supernatants are removed and transferred to the MSD plates.Fresh samples are typically tested, although they may be maintained at−30 to −70° C. until analysis.

Interferon-α and Tumor Necrosis Factor-α Analysis

MSD MULTI-SPOT plates contain within each well capture antibodies forhuman TNF-α and human IFN-α that have been pre-coated on specific spots.Each well contains four spots: one human TNF-α capture antibody (MSD)spot, one human IFN-α a capture antibody (PBL Biomedical Laboratories,Piscataway, N.J.) spot, and two inactive bovine serum albumin spots. Thehuman TNF-α capture and detection antibody pair is from MesoScaleDiscovery. The human IFN-α multi-subtype antibody (PBL BiomedicalLaboratories) captures all IFN-α subtypes except IFN-α F (IFNA21).Standards consist of recombinant human TNF-α (R&D Systems, Minneapolis,Minn.) and IFN-α (PBL Biomedical Laboratories). Samples and separatestandards are added at the time of analysis to each MSD plate. Two humanIFN-α detection antibodies (Cat. Nos. 21112 & 21100, PBL) are used in atwo to one ratio (weight:weight) to each other to determine the IFN-αconcentrations. The cytokine-specific detection antibodies are labeledwith the SULFO-TAG reagent (MSD). After adding the SULFO-TAG labeleddetection antibodies to the wells, each well's electrochemoluminescentlevels are read using MSD's SECTOR HTS READER. Results are expressed inpg/mL upon calculation with known cytokine standards.

Assay Data and Analysis

In total, the data output of the assay consists of concentration valuesof TNF-α or IFN-α (y-axis) as a function of compound concentration(x-axis).

A plate-wise scaling is performed within a given experiment aimed atreducing plate-to-plate variability associated within the sameexperiment. First, the greater of the median DMSO (DMSO control wells)or the experimental background (usually 20 pg/mL for IFN-α and 40 pg/mLfor TNF-α) is subtracted from each reading. Negative values that mayresult from background subtraction are set to zero. Each plate within agiven experiment has a reference compound that serves as a control. Thiscontrol is used to calculate a median expected area under the curveacross all plates in the assay. A plate-wise scaling factor iscalculated for each plate as a ratio of the area of the referencecompound on the particular plate to the median expected area for theentire experiment. The data from each plate are then multiplied by theplate-wise scaling factor for all plates. Only data from plates bearinga scaling factor of between 0.5 and 2.0 (for both cytokines IFN-α,TNF-α) are reported. Data from plates with scaling factors outside theabove mentioned interval are retested until they bear scaling factorsinside the above mentioned interval. The above method produces a scalingof the y-values without altering the shape of the curve. The referencecompound used is2-[4-amino-2-ethoxymethyl-6,7,8,9-tetrahydro-α,α-dimethyl-1H-imidazo[4,5-c]quinolin-1-yl]ethanolhydrate (U.S. Pat. No. 5,352,784; Example 91). The median expected areais the median area across all plates that are part of a givenexperiment.

A second scaling may also be performed to reduce inter-experimentvariability (across multiple experiments). All background-subtractedvalues are multiplied by a single adjustment ratio to decreaseexperiment-to-experiment variability. The adjustment ratio is the areaof the reference compound in the new experiment divided by the expectedarea of the reference compound based on an average of previousexperiments (unadjusted readings). This results in the scaling of thereading (y-axis) for the new data without changing the shape of thedose-response curve. The reference compound used is2-[4-amino-2-ethoxymethyl-6,7,8,9-tetrahydro-α,α-dimethyl-1H-imidazo[4,5-c]quinolin-1-yl]ethanolhydrate (U.S. Pat. No. 5,352,784; Example 91) and the expected area isthe sum of the median dose values from an average of previousexperiments.

The minimum effective concentration is calculated based on thebackground-subtracted, reference-adjusted results for a given experimentand compound. The minimum effective concentration (μmolar) is the lowestof the tested compound concentrations that induces a response over afixed cytokine concentration for the tested cytokine (usually 20 pg/mLfor IFN-α and 40 pg/mL for TNF-α). The maximal response is the maximalamount of cytokine (pg/ml) produced in the dose-response.

The complete disclosures of the patents, patent documents, andpublications cited herein are incorporated by reference in theirentirety as if each were individually incorporated. Variousmodifications and alterations to this invention will become apparent tothose skilled in the art without departing from the scope and spirit ofthis invention. It should be understood that this invention is notintended to be unduly limited by the illustrative embodiments andexamples set forth herein and that such examples and embodiments arepresented by way of example only with the scope of the inventionintended to be limited only by the claims set forth herein as follows.

1. A compound of the Formula I

wherein: Y is selected from the group consisting of —C(O)— and —C(O)—O—;R is selected from the group consisting of alkyl, aryl, arylalkylenyl,heteroaryl, heteroarylalkylenyl, heterocyclyl, andheterocyclylalkylenyl; wherein aryl and arylalkylenyl are unsubstitutedor substituted by one or more substituents selected from the groupconsisting of alkyl alkoxy, aryl, and halogen; and wherein the atom inheterocyclyl attached to Y is a carbon atom; and R′ is selected from thegroup consisting of methyl, ethyl, and n-propyl; or a pharmaceuticallyacceptable salt thereof.
 2. The compound or salt of claim 1 wherein Y is—C(O)—.
 3. The compound or salt of claim 1 wherein Y is —C(O)—O—.
 4. Thecompound or salt of claim 1 wherein R is alkyl, aryl, or arylalkylenyl.5. The compound or salt of claim 1 wherein R is C₁₋₁₀ alkyl.
 6. Thecompound or salt of claim 1 wherein R is C₁₋₅ alkyl.
 7. The compound orsalt of claim 1 wherein R is selected from the group consisting ofmethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl.8. The compound or salt of claim 1 wherein R is aryl.
 9. The compound orsalt of claim 1 wherein R is phenyl.
 10. The compound or salt of claim 1wherein R is arylalkylenyl.
 11. The compound or salt of claim 1 whereinR is benzyl.
 12. The compound or salt of claim 1 wherein R′ is ethyl.13. The compound or salt of claim 1 wherein R′ is methyl.
 14. Thecompound or salt of claim 1 wherein R′ is n-propyl.
 15. A pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundor salt of claim 1 and a pharmaceutically acceptable carrier.
 16. Amethod of inducing cytokine biosynthesis in an animal comprisingadministering an effective amount of a compound or salt of claim
 1. 17.A method of treating a viral disease in an animal comprisingadministering a therapeutically effective amount of a compound or saltof claim
 1. 18. A method of treating a neoplastic disease in an animalcomprising administering a therapeutically effective amount of acompound or salt of claim 1.